CN104610086A - Stearoyl amino acid compound, and preparation method and applications thereof - Google Patents

Abstract

The invention discloses a stearyl amino acid compound, which has antioxidant activity, and its structure is shown in the following general formula (I), wherein, R ₁ represents H or C ₁ which may be substituted by one or more substituents _-6 linear or branched alkyl groups, the substituents are selected from: hydroxyl, amino, mercapto, carboxyl, aryl, amido, alkylthio and 5 or 7 with 1-2 nitrogen atoms R ₂ represents H or C _1-4 alkyl; R ₃ represents C _11-25 saturated or unsaturated aliphatic hydrocarbon group. The invention also discloses the preparation method and application of the stearyl amino acid compound. The stearoyl amino acid compound of the invention can effectively resist the oxidative damage of cells induced by glutamic acid, has remarkable antioxidant activity, can be used for preparing antioxidant drugs, and has very broad application prospects.

Description

Stearoyl amino acid compound and its preparation method and application

technical field

The invention relates to the technical field of medicine, in particular to a stearyl amino acid compound with antioxidant activity, a preparation method and application thereof.

Background technique

N-stearyl amino acids (NSAs) are amide compounds formed by combining the carboxyl group of stearic acid with the α-amino group of amino acid. As early as the 1950s and 1960s, there were literatures reporting its synthesis method. In recent years, there are also research reports on its pharmacological activity, but there is no research report on the antioxidant effect of such compounds.

(1) Synthesis method of NSAs

In the synthesis of NSAs, stearic acid is generally made into stearyl chloride to improve the activity of the acylation reaction; amino acid is reacted with hydrochloric acid-methanol to obtain amino acid methyl ester to protect the carboxyl group. Then stearyl chloride and amino acid methyl ester undergo acylation reaction in pyridine solution to generate N-stearyl amino acid methyl ester, which is then hydrolyzed to obtain NSAs (Zeelen, Havinga. Recl Trav Chim. 1958, 77, 267-271).

(2) Study on the pharmacological activity of NSAs

Antibacterial effect: These compounds have activity against Gram-negative bacteria (Staphylococcus aureus, Micrococcus luteum and Bacillus cereus), Gram-positive bacteria (Escherichia coli and Pseudomonas aeruginosa), Among them, N-stearyl proline has the strongest effect, and the activity of other compounds depends on the structure of amino acids. Generally speaking, aromatic NSA>acidic NSA>basic NSA (Sivasamy A, Krishnaveni M, Rao PG.J Am Oil Chim Soc. 2001, 78(9), 897-902).

Antiviral effect: These compounds can be used as non-N-acetylneuraminidase inhibitors of influenza virus neuraminidase (NA), and can effectively inhibit the activity of NA in a dose-dependent manner. Among a series of NSAs derivatives, N-hydroxytetradecyl-D-cysteine and N-tetradecyl-O-acetyl-D-serine have the strongest activity, and their mechanism of action is non-competitive inhibition of enzymes activity. Since this type of compound is not only a selective inhibitor of viral NA, but also effective for enzymes of various other viruses (except for enzymes of cholera type V and human placenta virus), it can be used as a lead for anti-influenza virus drugs Compounds (Kondoh, Mitsuyo, Furutani, et al. Biosci Biotechnol Biochem. 1997, 61(5), 870-874).

However, prior to the present invention, there have been no published reports of the compounds of the present invention being used for their antioxidant effects.

Contents of the invention

The technical problem to be solved by the present invention is to provide a stearoyl amino acid compound, which has antioxidant activity and can be used to prevent or treat damage or disease caused by oxidative toxicity.

In addition, it is also necessary to provide a preparation method and application of the stearoyl amino acid compound.

In order to solve the problems of the technologies described above, the present invention is realized through the following technical solutions:

In one aspect of the present invention, a stearyl amino acid compound with antioxidant activity is provided, the structure of which is shown in the following general formula (I):

Wherein, _R represents H or C _1-6 straight chain or branched alkyl that may be substituted by one or more substituents selected from: hydroxyl, amino, mercapto, carboxyl, aryl , amide group, alkylthio group and 5 or 7-membered heterocyclic group with 1-2 nitrogen atoms; R ₂ represents H or C _1-4 alkyl; R ₃ represents C _11-25 saturated or unsaturated aliphatic Hydrocarbyl.

The above-mentioned C _1-6 alkyl group refers to a straight chain or branched chain alkyl group having 1 to 6 carbon atoms. For example: methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, pentyl, neopentyl, hexyl. A linear or branched alkyl group having 1 to 4 carbon atoms is preferred, and a linear alkyl group having 1 to 2 carbon atoms is more preferred.

Among the above substituents, the hydroxyl group includes alcoholic hydroxyl group and phenolic hydroxyl group; the 5- or 7-membered heterocyclic group with 1-2 nitrogen atoms can be selected from imidazolyl, tetrahydropyrrolyl and indolyl.

The above-mentioned C _11-25 aliphatic hydrocarbon group refers to a saturated or unsaturated aliphatic hydrocarbon group with 11 to 25 carbon atoms, wherein the saturated aliphatic hydrocarbon group refers to a linear or branched alkyl or cycloalkyl group, such as dodecyl Alkyl, octadecyl, cyclododecyl, cyclooctadecyl, etc., unsaturated aliphatic hydrocarbon group refers to alkenyl, alkynyl or alkadienyl, alkenyl such as 1-dodecenyl , 2-dodecenyl, alkynyl such as 1-octadecynyl, 2-octadecynyl, alkadienyl such as 1,3-octadecenyl, 7,9-octadecenyl, etc. A linear or branched alkyl group having 17 to 25 carbon atoms is preferred, and a linear alkyl group having 17 carbon atoms is particularly preferred.

_R2 is preferably H.

Take the following compound as an example:

R ₁ is preferably an alkyl group with 1 to 2 carbon atoms substituted by hydroxyl, aryl, tetrahydropyrrolyl and indolyl, more preferably _R is substituted by alcoholic hydroxyl, phenolic hydroxyl, phenyl, tetrahydropyrrolyl and An alkyl group having 1 carbon atom substituted by an indolyl group, most preferably R is an alkyl group having ₁ carbon atom substituted by a phenolic hydroxyl group or an alcoholic hydroxyl group.

It is particularly preferred that _R1 is an alkyl group having 1 carbon atom substituted by a phenolic hydroxyl group or an alcoholic hydroxyl group, _R2 is H, _{and R3} is a linear alkyl group having 17 carbon atoms.

Preferred compounds of the present invention are:

N-Stearyl Tyrosine

In another aspect of the present invention, there is provided a method for preparing the above-mentioned stearyl amino acid compound, which method comprises the following formula (IV) compound

React with the following formula (Ⅲ) compound under alkaline conditions to prepare the general formula (I) compound

Preferably, the compound of formula (IV) is prepared by reacting the compound of formula (II) with coupling agent 1-hydroxybenzotriazole

Preferably, the compound of formula (II) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, stearic acid, triethylamine, in the catalyst 4-dimethyl Prepared under the action of aminopyridine.

Preferably, the compound of formula (III) is prepared by reacting amino acid with methanol hydrochloride.

In another aspect of the present invention, there is also provided a pharmaceutical composition, which contains a safe and effective amount of the stearyl amino acid compound of the general formula (I) above, and contains one or more pharmaceutically acceptable carriers .

The above-mentioned acceptable carriers are non-toxic, can assist administration and have no adverse effect on the therapeutic effect of the compound of general formula (I). Such carriers can be any solid, liquid, semisolid or, in aerosol compositions, gaseous excipients commonly available to those skilled in the art. Solid pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl stearyl, sodium chloride , Anhydrous skim milk, etc. Liquid and semisolid excipients can be selected from glycerin, propylene glycol, water, ethanol and various oils, including those derived from petroleum, animal, vegetable or synthetic oils, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc., preferably Liquid carriers, especially for injectable solutions, include water, saline, aqueous dextrose and glycol. In addition, other adjuvants such as flavoring agents and sweetening agents can also be added to the composition.

The compounds of the present invention are administered in a therapeutically effective amount, which can be administered orally, systemically (e.g., transdermally, nasally, or by suppository), or parenterally (e.g., intramuscularly, intravenously, or subcutaneously). ). The preferred mode of administration is oral, which can be adjusted according to the extent of the disease.

The actual amount of compound administered (i.e., the active ingredient) of the present invention depends on many factors, such as the severity of the disease being treated, the age and relative health of the subject, the potency of the compound used, the route and form of administration, and other factors. factor.

Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional methods in the field of pharmacy. For example, the compound (active ingredient) is mixed with one or more carriers, and then prepared into the desired dosage form, such as tablet, pill, capsule, semi-solid, powder, sustained-release dosage form, solution, suspension, Dosages, aerosols, etc.

In another aspect of the present invention, the application of the above compounds in the preparation of antioxidant drugs is also provided.

The experiment of glutamate-induced oxidative damage to PC12 cells and cortical neurons confirmed that the stearyl amino acid compound of the present invention can effectively resist glutamate-induced cell oxidative damage, and can almost completely reverse glutamate-induced cell oxidative damage The morphological changes indicate that the stearyl amino acid compound of the present invention has significant antioxidant activity, can be used to prepare antioxidant drugs, and has a very broad application prospect.

Description of drawings

The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments.

Fig. 1 is the histogram of non-caspase-dependent oxidative damage of PC12 cells induced by glutamate in Example 4 of the present invention;

Fig. 2 is the oxidative toxicity histogram and light microscope observation diagram of NsTyr of the embodiment 4 of the present invention against glutamate-induced PC12 cells;

3 is a histogram of non-caspase-dependent oxidative damage induced by glutamate in Example 5 of the present invention in primary cortical neurons;

4 is a histogram and light microscope observation diagram of NsTyr against glutamate-induced cortical neuron oxidative toxicity in Example 5 of the present invention;

Fig. 5 is a light microscope observation diagram of NsTyr in Example 6 of the present invention inhibiting glutamate-induced ROS accumulation in PC12 cells.

Detailed ways

The preparation of embodiment 1N-stearyl amino acid

(1) Protection of amino acids - carboxymethyl esterification

Amino acid reacts with hydrochloric acid-methanol to obtain amino acid methyl ester. Stearic acid and thionyl chloride are heated to reflux to obtain stearyl chloride.

Amino acid methyl ester was dissolved in pyridine, the above stearyl chloride was added dropwise, and the reaction was stirred overnight. After extraction, recrystallization, etc., stearyl amino acid methyl ester is obtained. Then it is hydrolyzed under alkaline conditions to obtain stearyl amino acid.

(2) Amino acid deprotection - first activation of stearic acid (active ester method)

Stearic acid (SA) reacts with N-hydroxysuccinimide (N-HOSu) to generate activated ester, and then reacts with amino acid compounds to obtain stearamide.

Stearic acid and N-hydroxysuccinimide were stirred and reacted for 16 hours under the action of dehydrating agent DCC, filtered and crystallized to obtain N-stearylsuccinimide (SA-OSu). Amino acid and SA-ONSu are stirred and reacted to obtain stearyl amino acid through acidification, extraction and crystallization.

The structure of the synthesized compound:

1. N-Stearyl Glycine Methyl Ester (NSGlyE)

2. Methyl N-stearyl glutamate (NSGluE)

3. N-Stearyl Phenylalanine Methyl Ester (NSPheE)

4. N-stearyl phenylalanine (NSPhe, DL-NSPhe)

5. N-Stearyl Proline (NSPro)

6. N-Stearyl Tyrosine (NSTyr)

7. N-Stearyl Cysteine (NSCys)

8. N-Stearyl Histidine (NSHis)

9. N-Stearyl Tryptophan (NSTrp)

10. N-Stearyl Lysine (NSLys)

11. N-Stearyl Serine (NESer)

12. N-Stearyl Leucine (NSLeu)

The preparation of embodiment 2N-stearyl amino acid

In this example, N-stearyl amino acid is prepared by a one-pot method. The one-pot method has mild reaction conditions, is not limited by amino acid solubility and system pH, and has ideal yield and purity. The following takes the one-pot preparation of N-stearyl tyrosine (NSTyr) as an example to describe the reaction steps of the one-pot method in detail.

At room temperature, with 1-hydroxybenzotriazole HOBT as coupling agent, 4-dimethylaminopyridine DMAP as catalyst, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride in One-pot synthesis of N-stearyl tyrosine methyl ester under alkaline conditions with pre-synthesized L-tyrosine methyl ester, which was hydrolyzed to N-stearyl tyrosine (NSTyr).

1. Preparation of Tyrosine Methyl Ester

One side of a 25ml two-necked bottle was connected to a drying tube, and the other was connected to a dropping funnel, and 5ml of methanol was added, and 0.8ml of acetyl chloride was added dropwise in an ice-salt bath, and stirred for 30 minutes to obtain methanol hydrochloride. Add 0.905g L-tyrosine and 5ml methanol to another 100ml round bottom flask. Stirring and adding methanol hydrochloride, after the solution is clarified, reflux for 24h. The reaction solution was dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dry the solvent to obtain L-tyrosine methyl ester hydrochloride as yellow flaky crystals. Add saturated sodium bicarbonate solution (20ml) to the solid until no more bubbles are produced, extract twice with dichloromethane (30ml), dry the organic phase, and evaporate to dryness to obtain 1.07g of a yellow solid of L-tyrosine methyl ester, the yield 95.6%.

2. N-Stearyl Tyrosine Methyl Ester (One Pot Method)

Add 1.704g (6mmol) of stearic acid, 1.22ml (9.8mmol) of triethylamine (EtB3BN) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride to a 250ml round bottom flask Salt 0.920g (4.8mmol), 4-dimethylaminopyridine (DMAP) 0.029g (0.24mmol), 1-hydroxybenzotriazole (HOBT) 0.648g (4.8mmol), L-tyrosine methyl ester 1.11g (4.8mmol) and 80ml of dichloromethane, stirred at room temperature for 24h, monitored by TLC. Add 1mol/L hydrochloric acid solution to the crude product to wash until white flocs no longer appear, extract 3 times with dichloromethane, and combine the organic phases; add saturated sodium bicarbonate solution to the above-mentioned collected organic phases to wash until no bubbles are produced, After extraction with dichloromethane three times, the organic phases were combined; the dichloromethane phase collected above was washed with saturated saline solution, dried over anhydrous sodium sulfate, and concentrated to obtain 1.4 g of a crude product. Silica gel column chromatography, developing solvent dichloromethane/methanol (4/1) to give L-stearyl tyrosine methyl ester, white powder 588.6mg, yield 82.7%, m.p.100-104°C. The product was added with 10ml of 1mol/L methanol-potassium hydroxide solution, heated to reflux at 70°C for 3h, and the clarified solution was refrigerated at 4°C for 24h. Filtrate to obtain a crude product, rinse with acetone 2-3 times, and dry to obtain 08.1 mg of white solid NsTyr-2K with a yield of 95.6%.

Example 3 Structure Identification

The structures of the stearyl amino acid compounds synthesized in Example 1 were identified by ¹ PHNMR and ¹³ PCNMR. The structural identification data of compounds 6 and 11 are listed below.

Compound 6:

White solid, mp, 106-108°C. 1HNMR (500MHz, CDCl3, TMS) δppm: 7.0 (2H, d, J=10.2Hz, H on the benzene ring); 6.7 (2H, d, J=10.5Hz, H on the benzene ring); 5.9 (1H, s, -NH-); 4.8 (1H, m, -CH-NH-); 3.1 (2H, m, -CH2-), 2.17 (2H, t, -CH2-CONH-); 1.4 (2H, m, -CH2-); 1.26 (28H, m, -(CH2)n-); 0.88 (3H, t, -CH3). 13CNMR (500MHz, CDCl3) δppm: 174.1 (-CONH-); 173.9 (-COOH); 155.3 (C of -OH on the benzene ring); 130.5, 130.3, 127.6, 121.8 and 115.8 (the other 5 C on the benzene ring ); 53.4 (-CH-NH-); 36.8 (-CH2-CONH-); 36.6 (-CH2-CHNH-); 32.0 (-CH2-); 31.6～22.6 (14×-CH2-); 14.0 (- CH3-).

The above data confirm that the compound is N-stearyl tyrosine.

Compound 11:

White powder, mp, 100～102℃. 1HNMR (300MHz, CD3OD, TMS) δppm: 4.48 (1H, t, -CH-NH-); 3.8 (2H, m, -CH2OH); 2.26 (2H, t, -CH2-CONH-); 1.62 (2H, m, -CH2-); 1.28 (28H, m, -(CH2)n-); 0.89 (3H, t, -CH3). 13CNMR (500MHz, CD3OD) δppm: 176.7 (-CONH-); 173.8 (-COOH); 63.3 (-CH2OH); 56.3 (-CH-NH-); 37.2 (-CH2-CONH-); 33.3 (-CH2- ); 31.1～24.0 (14×-CH2-); 14.7 (-CH3-).

The above data confirm that the compound is N-stearylserine.

Example 4 N-stearyl tyrosine (NsTyr) against glutamate-induced oxidative toxicity of PC12 cells

1. Experimental method

(1) Cell culture

Rat adrenal medulla pheochromocytoma PC12 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were grown in DMEM medium containing 10% fetal bovine serum and cultured at 37°C in a 5% CO ₂ incubator. The medium was changed every other day and a half, and the cells were passaged when they grew to 80%-90% confluence.

(2) Establishment of glutamate-induced PC12 cell injury model and drug treatment

The injury model was established by PC12 cells cultured for 24 hours, and the culture plate was taken out from the carbon dioxide incubator and treated with 10 mmol/L glutamate for 24 hours to establish the injury model. Different concentrations of the test drug NsTyr (white powdery solid, developed and synthesized by our teaching and research department, the purity was >98% by HPLC analysis, and DMSO was used to help dissolve it) was added 1 h before glutamate injury, and the drug existed throughout the injury process.

(3) MTT detection

Cells were inoculated at a density of 3×10 ⁴ in 96-well culture plates and then damaged by glutamate for 24 hours. During this period, different drugs were added according to the needs of the experiment. After the reaction, add 10 μl of MTT (10 mg/ml, Sigma Company), incubate at 37°C for 4 hours, discard the supernatant, add 100 μl of DMSO, shake for about 10 minutes until the crystals are completely dissolved, and read the absorbance at 490 nm with a microplate reader. At the same time, set zero wells (medium, DMSO, MTT) and control wells (cells, culture medium, drug dissolution medium of the same concentration, DMSO, MTT), set 6 replicate wells for each group, and repeat the independent experiment 3 times. Because succinate dehydrogenase in living cells catalyzes MTT to generate colored crystal products, the absorbance value reflects cell viability, and the ratio of the average absorbance value of the treatment group to the untreated control group represents the relative change in cell viability.

(4) TUNEL staining

Deoxyribonucleotide terminal transferase-mediated nick end labeling (TUNEL staining) uses chromosomal DNA double-strand breaks or single-strand breaks to generate a large number of sticky 3'-OH ends, which can be transferred at the end of deoxyribonucleotides Under the catalysis of enzyme (TdT), deoxyribonucleotides and fluorescein are labeled to the 3'-OH end of DNA, so as to detect apoptotic cells. Seed PC12 cells at a concentration of 5×10 ⁵ cells/ml in a 24-well plate covered with coverslips. After drug treatment, wash off the culture medium with PBS, add 1ml of 4% paraformaldehyde to each well, fix for 20min, PBS Wash 3 times. Permeabilize the membrane with 0.1% Triton-X100 on ice for 10 minutes. Incubate TUNEL staining solution (Reagent 1 and Reagent 2 at a ratio of 1:9 on ice, TUNEL kit purchased from Invitrogen) for 90 min at room temperature in the dark. The slides were sealed with anti-fluorescence quenching mounting solution, observed and photographed with an optical microscope. Five fields of view were selected for each coverslip, and the nuclei positive for TUNEL staining were red, representing apoptotic cells in the field of view.

2. Experimental results

(1) Glutamate induces caspase-independent oxidative damage in PC12 cells

After PC12 cells were treated with 10mmol/L glutamic acid for 24h, cell viability was detected by MTT method. As shown in Figure 1A, the viability of PC12 cells was significantly reduced (P < 0.001).

PC12 cells were pretreated with glutamate receptor antagonists (5 μmol/L MK-801, 20 μmol/L CNQX) or antioxidants (5 mmol/LNAC) for 1 hour, and then added with 10 mmol/L glutamic acid for 24 hours, MTT method The cell viability was detected, and the results showed that the PC12 cell viability in the antioxidant group was significantly higher than that in the glutamate group (P<0.001), while the cell viability in the receptor antagonist group was not different from that in the glutamate group (P>0.05).

In addition, different concentrations of caspases inhibitor z-VAD-FMK were pretreated for 1 hour, and then 10 mmol/L glutamic acid was added for 24 hours. The cell viability was detected by MTT method, and different concentrations of caspases inhibitors had no significant protection against PC12 cell damage. effect (P>0.05) (Fig. 1B). These results suggest that glutamate mainly induces PC12 cell injury through oxidative toxicity, rather than excitotoxicity mediated by glutamate receptors; at the same time, it suggests that glutamate-induced oxidative toxicity in PC12 cells is mediated by a caspase-independent pathway.

(2) NsTyr can resist glutamate-induced oxidative toxicity in PC12 cells

0-20μmol/L NsTyr pretreated PC12 cells for 1h, and then added 10mmol/L glutamate for 24h. MTT results showed that NsTyr could resist glutamate-induced oxidative damage in a dose-dependent manner (1-10μmol/L, Fig. 2A), and 10 μmol/L NsTyr had the strongest protective effect (P<0.001). Light microscope observation revealed that 10 μmol/L NsTyr could almost completely reverse the morphological changes induced by glutamate (Figure 2B). Using TUNEL staining method, it was found that the number of TUNEL-positive cells significantly increased after 24 hours of glutamic acid treatment, and 10 μmol/L NsTyr pretreatment could significantly reduce this increase (Fig. Acid-induced oxidative damage.

Example 5 NsTyr against glutamate-induced oxidative toxicity of cortical neurons

1. Experimental method

(1) Establishment of glutamate-induced injury model of primary cortical neurons and drug treatment

After anesthetizing SD rats at 14-16 days pregnant with ether, disinfect and remove the fetal rats by laparotomy, decapitate them on ice, place them in the dissection solution D-Hank's solution, separate the cerebral cortex, peel off the meningeal blood vessels, collect enough cortex, and cut it into pieces Make it into a paste shape, transfer it to a 15ml centrifuge tube with a pipette gun, let it stand still, wait for the cortex to settle, remove the dissection fluid as much as possible, add an appropriate amount of trypsin digestion solution according to the amount of the cortex (0.25% trypsin 1ml, dilute to 5ml with DMEM), Incubate at 37°C for 8 min. Gently pipette until the cells are evenly dispersed. Add a small amount of fetal bovine serum to stop the digestion, centrifuge at 1000g for 5min, and discard the supernatant. Add DMEM containing 10% fetal bovine serum and 5% horse serum to the cell pellet, and pipette evenly. After counting the cells, adjust the cell density and inoculate on the culture plate pre-coated with poly-lysine. After culturing in the cell incubator at 37°C for 24 hours, they were cultured in NeuroBasal medium + 1% B27 nutrient solution. On the third day of culture, the neurons were removed from the CO2 incubator for glutamate injury. Different concentrations of the test drug NsTyr (white powdery solid, developed and synthesized by our teaching and research department, the purity was >98% by HPLC analysis, and DMSO was used to help dissolve it) was added 1 hour before glutamate injury, and the drug acted on the entire injury process .

2. Experimental results

(1) Glutamate induces caspase-independent oxidative damage in primary cortical neurons

The primary cortical neurons cultured for 3 days were treated with 1mmol/L glutamic acid for 24 hours, and the cell viability was detected by the MTT method. As shown in Figure 3, the neuron viability was significantly reduced (P<0.001).

If neurons were pretreated with glutamate receptor antagonists (5 μmol/L MK-801, 20 μmol/L CNQX), antioxidants (5 mmol/L NAC) or caspases inhibitors (20 μmol/L z-VAD-FMK) After 1 hour, 1mmol/L glutamic acid was added for 24 hours, and the cell viability was detected by MTT method. There was no difference in the cell viability of the glutamate group and the caspase inhibitor group compared with the glutamate group (P>0.05). These results suggest that glutamate induces neuronal damage through oxidative toxicity, and glutamate-induced neuronal oxidative toxicity is mediated by a caspase-independent pathway.

The damage mechanism of glutamate to neurons is similar to that of PC12 cells.

(2) NsTyr against glutamate-induced oxidative toxicity of cortical neurons

0-20μmol/L NsTyr pretreated cortical neurons for 1h, and then added 1mmol/L glutamate for 24h. MTT results showed that NsTyr could resist glutamate-induced oxidative damage in a dose-dependent manner (5-10μmol/L , Fig. 4A), and 10 μmol/L NsTyr had the strongest protective effect (P<0.001). Light microscope observation revealed that after 24 hours of glutamate treatment, neuron synapses ruptured, and some cell bodies shrank or even floated, while 10 μmol/L NsTyr could almost completely reverse the morphological changes of neurons induced by glutamate (Figure 4B).

Comparing the experimental results of Example 4 above, it can be seen that the activity of NsTyr against glutamate-induced oxidative toxicity of cortical neurons is similar to its corresponding activity on PC12 cells.

Example 6 NsTyr inhibits glutamate-induced ROS accumulation in PC12 cells

1. Experimental method

(1) Detection of intracellular ROS (reactive oxygen free radicals) level

DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) is a non-fluorescent non-polar substance that can pass through the cell membrane and be oxidized by ROS in the cell to the fluorescent substance DCF (2',7'-dichlorofluorescein), so the DCF Fluorescence intensity represents the level of ROS in the cell, which can be directly detected with a fluorescence microscope. PC12 cells were seeded on glass slides at a density of 2×10 ⁶ /well. After glutamate damage for 6 hours and 8 hours, 5 μl of 10 μmol/L DCFH-DA was added to each well, incubated at 37°C for 30 minutes, the culture medium was discarded, washed 3 times with PBS, and used Fluorescence microscope was used to observe and take pictures (the wavelength of excitation light is 488nm, and the wavelength of emission light is 525nm), and the image J software calculated the DCF fluorescence intensity value.

2. Experimental results

ROS accumulation is a key upstream event in glutamate-induced oxidative damage. The H2DCF-DA fluorescent probe was used to detect the ROS levels in each group of cells at different time points. As shown in Figure 5, after 8 hours of glutamate treatment, the level of intracellular ROS was significantly higher than that of the CON group (P<0.001vs.CON), and after NsTyr pretreatment, the above experiment was repeated, and the level of ROS induced by glutamate increased was significantly inhibited (P<0.001vs.GLU).

The above-mentioned embodiments only express the implementation manner of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (9)

1. have a stearyl amino-acid compound for anti-oxidant activity, this compound structure is as shown in following general formula (I):

Wherein, R ₁the C representing H or can be replaced by one or more substituting group _1-6straight chain or alkyl with side chain, described substituting group is selected from: hydroxyl, amino, sulfydryl, carboxyl, aryl, amide group, alkylthio and have 5 or 7 yuan of heterocyclic radicals of 1-2 nitrogen-atoms;

R ₂represent H or C _1-4alkyl;

R ₃represent C _11-25saturated or unsaturated aliphatic hydrocarbyl moiety.

2. compound according to claim 1, is characterized in that, described R ₁for the C that can be replaced by 1-3 substituting group _1-4straight chain or alkyl with side chain, described substituting group is selected from: hydroxyl, amino, sulfydryl, carboxyl, aryl, amide group, alkylthio and have 5 or 7 yuan of heterocyclic radicals of 1-2 nitrogen-atoms; R ₂for H; R ₃for C ₁₇straight chain saturated alkyl.

3. compound according to claim 2, is characterized in that, described compound is the NSTyr of following structural formula:

4. the preparation method of stearyl amino-acid compound described in claim 1, is characterized in that, the method comprises lower formula IV compound

Obtained general formula (I) compound is reacted in the basic conditions with lower formula III compound

5. preparation method according to claim 4, is characterized in that, described formula IV compound is reacted by lower formula II compound and coupler I-hydroxybenzotriazole to prepare

6. preparation method according to claim 5, it is characterized in that, described formula II compound is by 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate, stearic acid, triethylamine, reacts and be prepared under the effect of catalyzer DMAP.

7. preparation method according to claim 4, is characterized in that, described formula III compound is reacted by amino acid and hydrochloric acid methanol to prepare.

8. a pharmaceutical composition, is characterized in that, said composition comprises the compound according to claim 1 of safe and effective amount and pharmaceutically acceptable carrier.

9. compound according to claim 1 is preparing the application in anti-oxidation medicine.